Influence of the phospholipid n-6:n-3 polyunsaturated fatty acid ratio on the oxidative phosphorylation of isolated cardiac mitochondria (part 4)
Respiration measurements: The mitochondrial oxygen consumption was measured at 37 C in a 1.2 mL respiration chamber with a Clark oxygen electrode in a respiration medium allowing the phosphorylation of ADP and the formation of creatine phosphate (CP). The medium contained sucrose (250 mM), tris-HCl (10 mM), KH2PO4 (10 mM), creatine (10 mM), N-acetylcysteine (10 mM), pH 7.4 and lipid-free bovine serum albumin (0.1%). The oxygen uptake was carried out in three buffers differing in their free calcium concentration. No EDTA was added in buffer 1. In buffers 2 and 3, EDTA was added at concentrations of 25 |jM and 1 mM, respectively. The pH was adjusted to 7.4. The respiration measurements were assessed using albumin-bound palmitoylcar-nitine (24 |j.M, palmitoyl-carnitine:albumin = 0.16) as oxidative substrate. Malate sodium (2.5 mM) was added to this substrate. The phosphorylation substrate was a mole to mole mixture of ADP (potassium salt) and magnesium acetate (formatting an ADP-Mg2+ complex).
The measurement chamber was totally filled with one of these respiration media. After the addition of the mitochondria (0.33 mg proteins/mL), the chamber was closed with a glass cap pierced with a hole fitted to a Hamilton syringe needle. After 10 s, the palmitoylcarnitine/malate mixture was added. After another 10 s interval, the ADP-Mg2+ complex was added at a final concentration of333 |j.M. The state III and state IVrespiration rates were determined according to the method of Chance and Williams. The ADP:O ratio was estimated according to the method of Estabrook. The biochemical integrity of the mitochondria was tested using glutamate (20 mM) as substrate in a conventional respiration medium without N-acetylcysteine, creatine and free calcium. In these conditions, the state III respiration rates (determined after the third ADP addition) were 256±10.3 and 282±17.9 ng atoms O2/min/mg for the FO and SSO mitochondria, respectively. The elevated respiratory control ratio (6.8±0.66 and 6.8±0.40 for the FO and SSO mitochondria, respectively) argued for a good mitochondrial integrity. This is a great opportunity for you to start saving some money instead of spending it all: you now have a perfectly reliable pharmacy you can from any time there is such a need and without being worried about the safety of your private information.